The present invention relates to an injectable physiologically acceptable aqueous fluorocarbon emulsion.
Certain fluorocarbon emulsions are known in the art, and their use in a number of medical applications has been described.
U.S. Pat. No. 3,911,138 is directed to an artificial blood comprising aqueous emulsions of perfluorocyclocarbons. The upper limit of the emulsion droplet size is given as 100 microns. The disclosure of this patent lacks any mention of sterilization procedures of the storage stability of these emulsions.
U.S. Pat. No. 3,958,014 relates to a process for making injectable emulsions of perfluorocyclocarbons. The preferred emulsion concentrations of perfluorocyclocarbon and lecithin are 25-30% (w/v) and 3/5% (w/v), respectively. Sterilization of the emulsion is performed in a rotating autoclave at 110xc2x0-120xc2x0 C. While the emulsion droplet size is in the range of 0.05-0.25 microns, the emulsion are stable for only two days.
U.S. Pat. No. 3,962,439 is related to emulsions of a group of fluorocarbons. The emulsifying agents are mixtures of phospholipids and fatty acids.
U.S. Pat. No. 3,989,843 discloses preparing fluorocarbon emulsions. Lecithin is not disclosed as being acceptable for use as an emulsifying agent. The emulsions of this patent, which are sterilized while being stirred, separate after being stored for several months.
U.S. Pat. No. 4,423,077 described compositions comprising stable emulsions of fluorocarbons 30-75% (w/v) and an emulsifying phospholipid, such as lecithin, 7-9% (w/v).
U.S. Pat. No. 4,252,827 describes emulsions consisting of F-Decalin and F-Tripropylamine mixtures which are sterilized in a rotary autoclave. When stored for six months at a temperature of 4xc2x0 C., the mean particle size of these emulsions was substantially unchanged.
U.S. Pat. No. 4,497,892 relates to emulsion compositions containing two perfluoro-compounds, 10-50% (w/v) total, a mixed emulsifying agent which comprises nonionic surfactants, phospholipids and fatty acids. The emulsions of this patent are sterilized in a rotary autoclave. The components are frozen and stored separately. The emulsions must be used within twenty-four hours of thawing and mixing the components.
U.S. Pat. Nos. 4,591,593 and 4,713,459 disclose processes for preparing F-N-methyldecahydroquinoline. An emulsion can be prepared by using lecithin as an emulsifying agent. Thermal sterilization is performed by using a rotary autoclave.
U.S. Pat. Nos. 4,865,836, 4,981,691, and 4,987,154, are directed to methods for making and using fluorocarbon emulsions.
JP 60-166,626 is directed to a process for making stable vascular contrast agent emulsions which contain fluorocarbons that have at least one bromine substitutent, and alpha-tocopherol (Vitamin E).
xe2x80x9cProperties of Polyorganosiloxane Surfaces on Glassxe2x80x9d, by M. J. Hunter et al., Industrial and Engineering Chemistry, Vol. 39, No. 11 (November 1947), discusses applying an organosilcone film upon a glass surface.
The disclosure of each of the above-identified references is hereby incorporated by reference.
The present invention relates broadly to a method for preparing an emulsion wherein the quantity of free or unemulsified fluorocarbon is minimized. Without wishing to be bound by any theory or explanation, it is believed that the quantity of free fluorocarbon within an emulsion can be substantially completely eliminated by reducing, if not preventing, any interaction between the emulsion and the interior surface of a storage container. For example, it is believed that pretreating the storage container causes formation of an interior monolayer coating which can prevent such interaction. Should a fluorocarbon emulsion be introduced or injected into a body, the presence of free fluorocarbon is undesirable because free fluorocarbon may cause formation of emboli in the bloodstream.
One aspect of the present invention relates to a sterilized emulsion which can be stored under ambient conditions in sealed infusion bottles for a year or more without significant deterioration, e.g., when stored at about 24xc2x0 C. the average emulsion droplet size increases to less than about 0.60 micron. Such an emulsion would be partially valuable for emergency use at facilities which are limited or over extended, for example, in disaster relief.
In another aspect, the present invention relates to a process for preparing perfluorocarbon (PFC) emulsions in physiologically compatible saline solutions which can be stored for lengthy periods, e.g., storage for more than about 2 years at a temperature of about 4xc2x0 C. or at least about 3 months at a temperature of about 24xc2x0 C. The quality of the emulsion can be improved by pretreating the storage containers, bottles, vials, among others. Typically, greater than about 99.8 wt. % of the PFC emulsified droplets remain in the size range of about 0.2-0.4 micron, when stored for a period longer than about one year at room temperature in a non-oxidizing atmosphere within, for example, sealed bottles.
The emulsions comprise about 10 through about 50% volume/volume (v/v) of at least one liquid perfluorocarbon (PFC), which has a molecular weight in the range of about 460-520, about 1-8% weight/volume (w/v) of at least one emulsifying agent, and the balance comprising a physiologically acceptable saline solution.
A sterilization emulsion, which is prepared by the method described herein, can be stored at ambient temperatures in sealed infusion bottles for at least about one year. The substantially complete elimination of any free fluorocarbon from the present emulsions allows such emulsions to be used safely on demand for medical applications. As a result, the present invention is particularly valuable for medical emergencies, and in situations wherein the availability of hospital equipment is limited.
The present invention relates broadly to minimizing the presence of free fluorocarbon in an emulsion. By minimizing the presence of free fluorocarbon, the invention can be employed as a process for preparing aqueous perfluorocarbon (PFC) emulsions which can be used in medical applications. By xe2x80x9cperfluorocarbonxe2x80x9d it is meant a substantially fluorinated fluorocarbon, e.g., this term encompasses completely fluorinated fluorocarbons and hydrogen-containing fluorocarbons. Further, such emulsions are stable over a period of at least one year when stored at room temperature (24xc2x0 C.), or for at least about 2 years when stored at about 4xc2x0 C. By xe2x80x9cstablexe2x80x9d it is meant that the droplet size of the emulsion does not increase significantly, e.g., when stored at about 4xc2x0 C. the average droplet size of the emulsion remains less than about 0.60 micron. Such emulsion are typically physiologically acceptable to the human body so that these emulsions can be employed for medical purposes.
Physiologically acceptable PFC emulsions have the ability to dissolve large volumes of gases within the human body such as oxygen and carbon dioxide. This ability enables acceptable PFC emulsions to be used for blood substitutes, and in medical treatments which are more effective when supplementary oxygen can be delivered to critical body organs such as the heart, brain, liver, kidneys, among other organs. In view of the world-wide shortage of human blood for use in transfusions, and increasing concern about its freedom from undesirable species, there is a long felt need for an artificial blood which is stable under ambient conditions, and free from infectious agents.
In addition to being effective blood substitutes, the emulsions prepared by the invention are medically useful in coronary angioplasty, cancer radiotherapy and chemotherapy, heart reperfusion, emergency treatment for stroke, among other uses. These emulsions also may be incorporated into a synthetic cerebrospinal fluid composition. For example, the PFC emulsion can be employed in acute stroke therapy by incorporating the emulsion within an oxygenated fluorocarbon-based nutrient emulsion which is administered by ventriculocisternal perfusion. In some cases, a fluorocarbon emulsion can be employed as an artificial cerebrospinal fluid (CSF), which is delivered by direct flow into the lateral ventricle of the brain. Upon effective delivery of the oxygenated fluorocarbon emulsion, the fluorocarbon emulsion may be capable of salvaging significant quantities of brain tissue.
The emulsions made by the process of this invention comprise about 10 through about 50% (v/v) of at least one liquid PFC which has a molecular weight in the range of about 460-520, and 1-8% (w/v) of at least one emulsifying agent, the balance being a physiologically acceptable aqueous solution of electrolytes. Normally, substantially completely all of the PFC becomes a component of the emulsion. The droplet size of the emulsion prior to sterilizing is about 0.10 micron. After sterilizing the emulsion, the particle size of the emulsion droplets ranges from about 0.2 to about 0.4 micron. The emulsion droplet size can be measured by using a Coulter N4MD sub-micron particle analyzer.
The PFCs are substantially chemically insert, and have no known adverse effect upon human physiology. Suitable PFC characteristics are such that following delivery to the body, the PFC is substantially completely expelled from the body through the respiratory system. Any suitable PFC, which is readily excreted from the body, can be used for preparing an emulsion that has substantially no free fluorocarbon. Suitable PFCs can be produced by any process which avoids contamination with physiologically unacceptable substances, or a process wherein such substances can be adquately removed by using conventional separation methods.
Specific examples of suitable PFCs are perfluorooctyl bromide (PFOB), bisperfluorobutyl ethylene (F-44E), and mixtures thereof, among others. A suitable PFC is encapsulated or emulsified by being contacted with at least one emulsifying agent such as a phospholipid, e.g., egg yolk lecithin.
The emulsion is present within an aqueous medium such as a dilute solution of salts. For example, the aqueous medium may comprise electrolytes which are present at concentrations that are sufficient to obtain, an isotonic emulsion. Typically, the aqueous electrolyte solution contains at least about 0.90 gram of electrolyte per liter of water for injection. Examples of suitable electrolytes comprise at least one member selected from the group of sodium chloride, potassium chloride, dibasic sodium phosphate, sodium bicarbanate, hydrated sodium citrate, hydrated calcium chloride, hydrated magnesium chloride, among others. For example, an aqueous electrolyte solution is obtained by preparing a buffered saline solution, e.g., about 7.4 g NaCl and 2.3 g NaHCO3 per liter. In some cases, the electrolyte solution comprises a modified Tyrode""s solution which has the following-general composition per liter:
The ingredients for a Tyrode""s solution can be dissolved into sterile water for injection, and diluted further to a final volume of about one liter.
For best results, the containers and equipment, which are used for preparing and storing the emulsion and its components, are thoroughly cleaned and sterilized prior to being used. Glassware is typically first cleansed by washing with aqueous isopropanol, e.g., about 70/30 v/v isopropanol/water; followed by rinsing with deionized water which has a neutral pH. Stainless steel parts of equipment, e.g., a homogenizer, which will contact the emulsion, can be washed at room temperature with an Alconox solution (Alcanox is a biodegradable compounded alkyl aryl sodium sulfonate available from Alconox Inc., New York, N.Y.). The glass equipment can then be heated in an oven to a temperature of at about 250xc2x0 C. to ensure that the glass equipment is substantially pyrogen free. Failure to effectively clean containers and other processing equipment may introduce contaminants into the emulsion which impair the utility of the final emulsion.
When a Tyrode""s solution is used as an electrolyte for preparing an emulsion, there can be a tendency for calcium carbonate to precipitate, thereby destabilizing the emulsion. For best results, calcium carbonate precipitation is reduced, if not prevented, by purging a freshly prepared electrolyte solution with carbon dioxide for about 15 to 30 minutes, and filtering the purged solution through an approximately 0.2 micron filter in a manner which assures sterility.
An electrolyte solution, which possesses an enhanced product sterility and a lower endotoxin content, can be obtained by conducting all the processing steps within a laminar flow hood. A class 100 laminar flow work space is normally satisfactory for this purpose. The laminar flow work space ensures that most particulate material above about 0.3 micron in size is continuously removed by filters, thereby providing a working atmosphere which contains less than about 100 particles above 0.3 micron per cubic foot. When the laminar flow work space is used in conjunction with conventional sterile processing techniques, the preparation of sterile, low endotoxin emulsions is enhanced.
For best results, prior to preparing the emulsion, the emulsifying agent, e.g., egg yolk lecithin, should be stored under nitrogen with dry ice refrigeration. Such storage is useful to prevent the emulsifying agent from undergoing any significant oxidative degradation, and/or microbial contamination. Oxidation typically has a detrimental effect on the stabilizing ability of the emulsifying agent.
The emulsion preparation process is begun by dispersing of intermixing the electrolyte solution and the emulsifying agent. The emulsifying agent can be dispersed within an electrolyte solution at room temperature by using a homomixer, e.g., supplied by Eppenbach, Greerco, Baldor/Boehm. The homomixer functions to apply a shear force or agitate the emulsion, thereby admixing the electrolyte and emulsifying agent to create an electrolyte/emulsfying agent dispersion which has a relatively small droplet size. Dispersing the emulsifying agent into the electrolyte solution typically produces a milky electrolyte/emulsifying agent dispersion. The dispersion can be heated to about 55xc2x0-60xc2x0 C. while under a nitrogen atmosphere, and then homogenized by using a Microfluidizer (supplied by Microfluidics, Inc.), or a Manton-Gaulin homogenizer, thereby producing a substantially translucent dispersion. The translucent dispersion is typically cooled to about 15xc2x0-20xc2x0 C. The average size of the dispersion particles or vesicles, which can be determined by using a Coulter N4MD sub-micron particle size analyzer, typically ranges between about 0.08-0.1 micron.
Prior to introducing the PFC into the translucent dispersion described previously, the PRC should be purged with carbon dioxide for about 30 minutes to ensure that substantially no calcium carbonate is precipitated in the emulsion, e.g., all of the calcium carbonate, if any, is converted to a soluble calcium bicarbonate. After purging the PFC with carbon dioxide, the PFC can be added slowly to the translucent dispersion while rapidly agitating the dispersion and maintaining the temperature at about 15xc2x0-20xc2x0 C. (an Eppenbach Homomixer is effective for agitating the dispersion.) An emulsion is usually obtained in about 15 minutes. The emulsion can be homogenized by being passed five to ten times through a Microfluidizer, or a Manton-Gaulin homogenizer. The emulsion can also be filtered by using a 10-12 micron filter to remove coarse particles.
The filtered emulsion is ready for storage, e.g., within glass infusion bottles. The presence of free fluorocarbon within the emulsion is substantially completely avoided, if not prevented, by pretreating the storage bottles or containers. For example, when storing the emulsion within a glass infusion bottle, the presence of free fluorocarbon within the emulsion can be prevented by pretreating the interior surface of the bottle. The interior of the bottles can be pretreated by being coated or sprayed at room temperature with, for example, a saline lecithin dispersion. The pretreated bottles can be inverted to drain the pretreating dispersion or solution, and then filled with the PFC emulsion. After being filled with the PFC emulsion, the bottles are typically back-filled or purged with nitrogen, and sealed.
In one aspect of the invention, the storage containers may be pretreated with a medical grade of silicone oil, e.g., Dow-Corning medical grade silicone oil no. 360. For example, the interior surface of a storage bottle is coated with silicone by filling the bottles With  with silicone oil. After draining the silicone oil, the bottles can be depyrogenated by baking in an oven at a temperature of about 250xc2x0 C. Without wishing to be bound by any theory or explanation, it is believed that pretreating the storage containers causes formation of a firmly bound polymeric monolayer on the glass surface which reduces the interaction between the bottle and the emulsion, thereby avoiding, if not completely preventing, the presence of free PFC. For example, it is believed that a surface coating of silicone oil may react with the interior glass surface of an infusion bottle, thereby forming a non-extractable silicone-containing monolayer which minimizes the interaction between the emulsion and the bottle. While particular emphasis has been placed upon using a lecithin dispersion and silicone oil for pretreating the emulsion containers, any pretreating fluid may be employed which does not adversely effect the utility of the emulsion. However, when the emulsion is employed for medical purposes, the pretreating fluid must be physiologically acceptable.
Another advantageous result which is obtained by pretreating the bottles is that should the presence of free fluorocarbon be detected, the free fluorocarbon can be substantially completely re-emulsified by agitating or shaking the container or bottle.
The sealed emulsion-containing bottles can be sterilized by any suitable method which does not adversely affect the emulsion. For example, a rotary or stationary autoclave, e.g., which is operated at a temperature of about 121xc2x0 C., can be used for achieving an acceptable Lethality Factor of about FO21.5. Lethality Factor is discussed in xe2x80x9cDisinfection, Sterilization, and Preservationxe2x80x9d, edited by Seymour S. Block, second edition, 1977; the content of which is incorporated by reference. In other words, the sealed bottles are heated in a manner which is capable of providing a quantity of heat that is equivalent to being exposed to a temperature of about 121xc2x0 C. for about 21 minutes.
When the emulsions of the invention are stored under ambient conditions, the emulsions are normally stable for at least about one year. However, the useful shelf life of the emulsions can be extended further by refrigerating the emulsion at temperatures no lower than about 4xc2x0 C.
Certain aspects of the invention are demonstrated in the following Examples. It is understood that these Examples are provided to illustrate, not limit, the scope of the appended claims.